Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a hydroxylation detection method of GLP-1 or a variant thereof, which is characterized by comprising the following steps:
mixing target analyte GLP-1 or a variant thereof with protease in a solvent, and performing enzyme digestion reaction to obtain polypeptide with a sequence shown in SEQ ID NO. 1, wherein the protease is selected from any one or two of Trypsin enzyme and Lys-C enzyme;
and (3) carrying out liquid chromatography or liquid-mass spectrometry analysis on the product of the digestion reaction.
Wherein the variant of GLP-1 is a polypeptide formed by adding, deleting or replacing amino acids on the basis of GLP-1, and the variant of GLP-1 is reserved with a polypeptide fragment with a sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3. And in the variant of GLP-1, the first amino acid linked to the C-terminus of the polypeptide fragment shown in SEQ ID NO. 2 or SEQ ID NO. 3 is not proline, otherwise the protease cannot recognize and cleave.
SEQ ID NO. 1 is EFIAWLVK.
SEQ ID NO. 2 is KEFIAWLVK.
SEQ ID NO. 3 is REFIAWLVK.
Wherein Lys-C cleaves KEFIAWLVK only.
The Trypsin enzyme may cleave XEFIAWLVK, and X may be K or R.
The term "variant" as used herein is a polypeptide that differs from the reference polypeptide, respectively, but retains essential properties (e.g., retains the biological activity of the reference polypeptide). A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Typically, the differences are limited, which makes the sequences of the reference polypeptide and the variant very similar in whole, and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more of amino acid substitutions, additions, deletions, or any combination. The substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code. Variants of the polypeptide may be naturally occurring, e.g., allelic variants, or they may be non-known naturally occurring variants. Non-naturally occurring variants of the polypeptide may be prepared by mutagenesis techniques or by direct synthesis. Variants may also include, but are not limited to, polypeptides or fragments thereof having one or more chemical modifications of its amino acid side groups. Chemical modifications include, but are not limited to: adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications of the amino acid side groups include, but are not limited to: acylation of lysine-epsilon-amino groups; n-alkylation of arginine, histidine or lysine; alkylation of glutamic acid or aspartic acid carboxylic acid groups; deamidation of glutamine or asparagine. Modification of the terminal amino group includes, but is not limited to: deamination, N-lower alkyl, N-di-lower alkyl and N-acyl modification. Modification of terminal carboxyl groups includes, but is not limited to: amide, lower alkylamide, dialkylamide and lower alkyl ester modifications. In addition, one or more pendant or terminal groups may be protected by protecting groups known to those of ordinary skill in protein chemistry.
As used herein, a "fragment" when used in reference to a polypeptide is a polypeptide having an amino acid sequence that is partially identical (but not fully identical) to the amino acid sequence of the entire naturally occurring polypeptide. Fragments may be "independent" or contained within a larger polypeptide, wherein they form portions or regions as a single contiguous region within a single larger polypeptide. Thus, fragments of GLP-1 may be "independent," or may be genetically fused and part of a larger amino acid sequence. For example, a fragment of naturally occurring GLP-1 will include amino acids 7 to 36 of naturally occurring amino acids 1 to 36. Furthermore, fragments of a polypeptide may also be variants of naturally occurring partial sequences. For example, a fragment of GLP-1 comprising amino acids 7-36 of naturally occurring GLP-1 may also be a variant having an amino acid substitution within a partial sequence thereof, e.g., an Ala to Gly at position 8.
In some embodiments, the variant of GLP-1 has a polypeptide fragment as set forth in SEQ ID NO. 4 or SEQ ID NO. 5. And in the variant of GLP-1, the first amino acid linked to the C-terminus of the polypeptide fragment shown in SEQ ID NO. 4 or SEQ ID NO. 5 is not proline. A variant of GLP-1 having a polypeptide fragment as shown in SEQ ID NO. 4, e.g. Abirudin. Variants of GLP-1 having a polypeptide fragment as set forth in SEQ ID NO. 5, such as dolapride.
SEQ ID NO. 4 is HGEGTFTSDVSSYLEGQAAKEFIAWLVK.
SEQ ID NO. 5 is HGEGTFTSDVSSYLEEQAAKEFIAWLVK.
Other variants of GLP-1 may have polypeptide fragments having 97%, 94%, 90% or 87% sequence identity to the polypeptide shown in SEQ ID NO. 2 or SEQ ID NO. 3.
In some embodiments, the variant of GLP-1 is abilurtin. It has the following amino acid sequence: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.
In some embodiments, the variant of GLP-1 is dolapride. It has the following amino acid sequence:
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
the object of the present invention is to detect hydroxylation of lysine in GLP-1 or variants thereof. By studying GLP-1, two proteases, trypsin enzyme and Lys-C enzyme, are obtained through screening, and under proper enzyme digestion conditions, such as enzyme proportion, enzyme digestion time and temperature, polypeptide fragments of the XEFIAWLVK sequence (X identified by the Trypsin enzyme is K or R, and X identified by the Lys-C enzyme is K) can be identified, and the polypeptide fragments of the EFIAWLVK sequence are obtained through cleavage. The fragment obtained by cleavage contains target possibly hydroxylated lysine to be detected, and the sequence of the polypeptide fragment after cleavage is shortened relative to the GLP-1 or a variant thereof with the original length, so that the detection is carried out by taking the polypeptide fragment after cleavage as a reference to more accurately distinguish whether the lysine in the sequence is hydroxylated and the proportion of the hydroxylation. The method of the invention can be applied to monitor or detect the hydroxylation level of lysine of the GLP-1 fusion proteins Abirudin and dolapride. In addition, the processing method before detection is simple, the EFIAWLVK sequence obtained by cutting is used as a detection reference, disulfide bonds are not contained, and only the small fragment is needed to be measured, so that reduction treatment is not needed, and two proteases obtained by screening are easy to obtain, so that the cost is saved, the detection efficiency is improved, and the sample processing steps are simplified.
In the invention, the ratio of enzyme to protein sample and the enzyme digestion time can influence the size and the number of fragments obtained after enzyme digestion. The more fragments, the more broken, and the more hetero peaks are separated out by liquid phase analysis. When the fragments are too many, the analysis result is affected, because the fragment peaks may overlap with the target peaks, thereby affecting the accuracy of the result and increasing the difficulty of liquid phase separation. Thus, the ideal result of the cleavage treatment is that the fewer and the better the cut pieces, the shorter and the better the cut pieces containing the target amino acid, and the greater the difference in length between the cut pieces containing the target amino acid and the other cut pieces.
In some embodiments, the mass ratio of the protease to the target analyte is 1: (20-200).
Preferably, the mass ratio of the protease to the target analyte is 1: (20-100). Specifically, the ratio may be 1: 20. 1: 30. 1: 40. 1: 50. 1: 60. 1: 70. 1: 80. 1: 90. 1:100.
in some embodiments, the temperature of the cleavage reaction is 15-30 ℃, and the time of the cleavage reaction is 2-20 hours. The time of the preferable enzyme digestion reaction can be 5h to 16h, and specifically can be 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h and 16h.
Preferably, before the target analyte is mixed with the protease, the method further comprises the step of changing the liquid of the target analyte, and adjusting the pH value of the obtained solution after the liquid change to 7-9. The pH can be adjusted to 7, 7.5, 8, 8.5, 9. The liquid change is not an essential step, and if the sample itself is in this pH range, the liquid change can be omitted, but the liquid change is conducive to the sample being in a consistent solution environment.
In some embodiments, the variant of GLP-1 is abilurtin. The quantitative analysis method is liquid chromatography, wherein the mobile phase of the liquid chromatography comprises a mobile phase A and a mobile phase B, the mobile phase A is formic acid aqueous solution, the mobile phase B is formic acid acetonitrile solution, the volume percentage of formic acid in the mobile phase A is 0.1-1%, and the volume percentage of formic acid in the mobile phase B is 0.1-1%;
the liquid chromatograph adopts gradient elution, and the elution program of the gradient elution is as follows: 0-35 min, wherein the volume fraction of the mobile phase A is reduced from 100% to 40% along with the time increase; the volume fraction of the mobile phase A is reduced from 40% to 0 after being increased with time for 35-40 min; the volume fraction of the mobile phase A is 0 after 40 min-50 min; 50 min-50.1 min, wherein the volume fraction of the mobile phase A is increased from 0 to 100% along with the time; 50.1-60 min, wherein the volume fraction of the mobile phase A is 100%.
In some embodiments, the variant of GLP-1 is dolapride. The quantitative analysis method is liquid chromatography, wherein the mobile phase of the liquid chromatography consists of a mobile phase A and a mobile phase B, the mobile phase A is formic acid aqueous solution, the mobile phase B is formic acid acetonitrile solution, the volume percentage of formic acid in the mobile phase A is 0.1-1%, and the volume percentage of formic acid in the mobile phase B is 0.1-1%;
the liquid chromatograph adopts gradient elution, and the elution program of the gradient elution is as follows: 0-40 min, wherein the volume fraction of the mobile phase A is reduced from 99.9% to 60% along with the time increase; the volume fraction of the mobile phase A is reduced from 60% to 10% with time from 40min to 42 min; 42-47 min, wherein the volume fraction of the mobile phase A is 10%; 47-48 min, wherein the volume fraction of the mobile phase A is increased from 10% to 99.9% along with the time; 48-55 min, wherein the volume fraction of the mobile phase A is 99.9%. In other embodiments, the variant of GLP-1 is Abirudin or dolapride. The quantitative analysis method is liquid chromatography, wherein the mobile phase of the liquid chromatography comprises a mobile phase A and a mobile phase B, the mobile phase A is formic acid aqueous solution, the mobile phase B is formic acid acetonitrile solution, the volume percentage of formic acid in the mobile phase A is 0.1%, and the volume percentage of formic acid in the mobile phase B is 0.1%;
the liquid chromatograph adopts gradient elution, and the elution program of the gradient elution is as follows: 0-12.5 min, wherein the volume fraction of the mobile phase A is reduced from 100% to 50% as time increases; 12.5 min-12.51 min, the volume fraction of the mobile phase A is reduced from 50% to 10% along with the time increase; 12.51 min-22.5 min, wherein the volume fraction of the mobile phase A is 10%;22.5 min-22.51 min, wherein the volume fraction of the mobile phase A is increased from 10% to 100% over time; 22.51 min-32.5 min, wherein the volume fraction of the mobile phase A is 100%.
In some embodiments, the method of qualitative analysis is a liquid-mass analysis, the liquid phase analysis method is as described above, wherein the mass spectrometry conditions are as follows: the taper hole voltage is 20V-160V, the capillary voltage is 1kV-3.5kV, the taper hole airflow is 30L/h-70L/h, the desolvation airflow is 300L/h-800L/h, the ion source temperature is 80-125 ℃, the desolvation airflow temperature is 250-500 ℃, and the high-energy gradient of collision energy is 23-55 eV.
The mass spectrometry conditions can be used for either apride or dolapride.
In some embodiments, the liquid chromatography column is a C18 column. The C18 Column is preferably a Column which may be selected from Waters Acquity HSS T3 Column (2.1X105 mm or 2.1X100 mm). Analysis may also be performed using Waters Acquity CSH C Column (2.1X105 mm or 2.1X100 mm) reverse phase chromatography or other C18 reverse phase chromatography columns, such as Waters Acquity BEH C Column (2.1X150 mm) or the like. The flow rate of the mobile phase may be 0.2mL/min to 1mL/min.
In some embodiments, the mobile phase may have a flow rate of 0.2mL/min to 0.3mL/min.
Taking Abirudin as an example, the active part peptide fragment of GLP-1 is as follows:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGR
trypsin may bind the above peptide Duan Qiecheng:
HGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GR (polarity too large to be retained in the column)
HGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GR (polarity too large to be retained in the column)
Thereby achieving the separation effect.
Lys-C enzyme was also used for analysis of this fusion protein, which cleaves GLP-1 containing active peptide fragments:
HGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GRHGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GRDAHK (HSA fusion partial peptide fragment)
The following are specific examples.
EXAMPLE 1 Abirascine sample hydroxylation assay
The sample pretreatment steps are as follows:
(1) 100 μg of purified Abirudin sample (concentration about 1 mg/mL) was taken and the solution was exchanged to 0.1M NH using a Zeba Spin desalting column4 HCO3 In solution (pH. Apprxeq. 7.8), about 100. Mu.L of sample was obtained.
(2) 1. Mu.g/. Mu.L of Promega Trypsin Gold trypsin dissolved in ultrapure water was added to 1. Mu.L so that the ratio of enzyme to protein sample was 1:100, enzyme cutting for 6 hours at room temperature, adding 10 mu L of 10% formic acid to stop enzyme cutting reaction, centrifuging for 5 minutes at 13000 Xg, and taking a supernatant and bottling for sample application.
Analysis of the loaded 2 μl sample, UPLC elution using a Waters Acquity HSS T Column (2.1×150 mm) Column, eluent a phase: 0.1% formic acid-water, phase B: 0.1% formic acid-acetonitrile.
The elution gradient is as follows in table 1:
TABLE 1
| Time(min) | Flow Rate(mL/min) | A％ | B％ |
| 0 | 0.2 | 100 | 0 |
| 35 | 0.2 | 40 | 60 |
| 40 | 0.2 | 0 | 100 |
| 50 | 0.2 | 0 | 100 |
| 50.1 | 0.2 | 100 | 0 |
| 60 | 0.2 | 100 | 0 |
For Abirudin, the active part peptide fragment of GLP-1 is as follows:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGR
trypsin may bind the above peptide Duan Qiecheng:
HGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GR (polarity too large to be retained in the column)
HGEGTFTSDVSSYLEGQAAK
EFIAWLVK (target peptide fragment)
GR (polarity too large to be retained in the column)
Thereby achieving the separation effect.
The results of the LC-MS analysis are shown in FIG. 1. Fig. 1 (B) shows a liquid chromatogram, and fig. (a) shows a mass spectrum total ion flow chart. Wherein the substance with the retention time of the liquid phase diagram (B) of 24.65min corresponds to the substance with the retention time of 24.75min of the mass diagram (A), and is a hydroxylation modified EFIAWLVK peptide segment (the proportion is shown as 1:T2 in table 2); wherein the substance with retention time of liquid phase diagram (B) of 25.00min corresponds to substance 25.07min of mass spectrum (A), and is an unmodified EFIAWLVK peptide fragment (the proportion is shown as 1:T2 in table 2).
Calculated, this hydroxylation modification ratio is shown in table 2 below, accounting for approximately 8%.
TABLE 2
Mass spectrometry information:
capillary voltage: 3.00kV
Taper hole airflow: 50L/h
Taper hole voltage: 40V
Desolventizing gas stream: 600L/h
Ion source temperature: 120 DEG C
Desolventizing gas temperature: 400 DEG C
Experiment MSe
Ion source: ESI acquisition time: 2-45min
The functions are as follows: MSe
Scanning arrangement
Low mass to charge ratio (m/z): 100
High mass to charge ratio (m/z): 2000
Scanning time: 0.5s
Collision energy:
low energy: 6eV
High energy gradient: 23-55eV
Taper hole voltage setting: the method of use is set.
The peptide fragment MS2 ion fragment confirmation mass spectrum is shown in figure 2.
EXAMPLE 2 cleavage Condition analysis
Example 2 is essentially the same as example 1, except that the room temperature cleavage times are different. The room temperature was 298K, 25 ℃. Typically 25℃and 100kPa are used as laboratory standard conditions.
The results of the liquid phase analysis with the cleavage time at room temperature of 6 hours are shown in FIG. 3 (circled portions are the hydroxylated modified peptide EFIAWLVK and the unmodified target peptide EFIAWLVK).
FIG. 4 is a pattern obtained by a Buffer control of 6h at room temperature, no obvious visible peak is found for 5-35min, indicating that Trypsin does not undergo self-digestion reaction under the conditions, and no obvious interference is caused to sample specificity identification.
As shown in FIG. 5, the result of the liquid phase analysis with the cleavage time at room temperature of 16 hours shows that the number of the hetero peaks is more and more remarkable than that of FIG. 3.
EXAMPLE 3 degree Larufin sample hydroxylation assay
The sample pretreatment steps are as follows:
(1) 100. Mu.g of purified dolapride sample (concentration about 1 mg/mL) was taken and the solution was exchanged to 0.1M Tris-HCl solution (pH. Apprxeq. 7.8) using a Zeba Spin desalting column to give about 100. Mu.L of sample.
(2) 1. Mu.g/. Mu.L of Promega Trypsin Gold trypsin dissolved in ultrapure water was added to 1. Mu.L so that the ratio of enzyme to protein sample was 1:100, enzyme cutting overnight at room temperature for 16h, adding 10 mu L of 10% formic acid to stop enzyme cutting reaction, centrifuging at 13000 Xg for 5min, and bottling and loading the supernatant.
Analysis of the loaded 2 μl sample, UPLC elution using Waters Acquity BEH C Column (2.1x150mm) eluent phase a: 0.1% formic acid-water, phase B: 0.1% formic acid-acetonitrile.
Elution gradient is as follows table 3:
TABLE 3 Table 3
The main peptide fragment obtained after the Trypsin cleavage:
HGEGTFTSDVSSYLEEQAAK
EFIAWLVK (hydroxylated modified peptide fragment)
GGGGGGGSGGGGSGGGGSAESK (hydrophilic linker).
The results of the liquid phase analysis are shown in FIG. 6. The circled position is the hydroxylation modified peptide EFIAWLVK and the unmodified target peptide EFIAWLVK, which shows that the peptide can be applied to various common reversed phase C18 chromatographic columns.
Example 4 comparison of enzyme digestion costs
The comparative document CN110865129a discloses a method for detecting various modification levels in dolapride, an analysis method for various modification levels in dolapride based on the cleavage of immunoglobulin G degrading enzymes (Ides) of streptococcus pyogenes. The method is characterized in that the IdeS digestion needs to be carried out overnight and the reduction reaction needs to be carried out. The Ides enzyme is very expensive and very rare and not readily available for a very long time. And it is described in this patent that trypsin treatment of the sample is such that hydroxylation modification of the peptide of interest cannot be monitored.
The cost of the Ides enzyme to the Trypsin enzyme is now compared.
Promega brand Ides enzyme quotation (5000 units) is $1,089.00/Each. According to the following steps of 1:100, a bottle can be made with 100 samples, with an average of $11 per sample, about 66 yuan per sample.
Promega brand Trypsin enzyme quotation (100 μg, about 1500 units) is $156.90/Each. According to the following steps of 1:100 processing mode one bottle can take approximately 100 samples, with an average of $1.6 per sample, about 10 yuan per sample.
Comprehensively, the price of the trypsins is cheaper.
In view of the cleavage pH, the range of the trpsin is preferably 7.0 to 9.0, more preferably 7.5. The pH of Ides is 5.1-7.6, and the optimal range is 6.6. Both can be achieved by normal buffers without a clear advantage or disadvantage in comparison to pH.
The invention has the advantages compared with the comparison document that: 1. the pretreatment of the sample is simple and convenient, the time is short, and the steps of denaturation reduction and the like are not needed. 2. The enzymes used in the present invention are cheaper than the enzymes in the reference. 3. The analysis method can accurately measure the hydroxylation proportion and overcome the technical bias.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.
Sequence listing
<110> Momordica Charantia Biotech Co Ltd
CANTONBIO Co.,Ltd.
Foshan Pu Jin Bioisystech Co.,Ltd.
<120> method for detecting hydroxylation of GLP-1 or variant thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<400> 1
Glu Phe Ile Ala Trp Leu Val Lys
1 5
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 2
Lys Glu Phe Ile Ala Trp Leu Val Lys
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 3
Arg Glu Phe Ile Ala Trp Leu Val Lys
1 5
<210> 4
<211> 28
<212> PRT
<213> Artificial Sequence
<400> 4
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
20 25
<210> 5
<211> 28
<212> PRT
<213> Artificial Sequence
<400> 5
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
20 25